Loss of outer retinal neurons and circuitry alterations in the DBA/2J mouse.

PURPOSE: The DBA/2J mouse line develops essential iris atrophy, pigment dispersion, and glaucomatous age-related changes, including an increase of IOP, optic nerve atrophy, and retinal ganglion cell (RGC) death. The aim of this study was to evaluate possible morphological changes in the outer retina of the DBA/2J mouse concomitant with disease progression and aging, based on the reduction of both the a- and b-waves and photopic flicker ERGs in this mouse line. METHODS: Vertically sectioned DBA/2J mice retinas were evaluated at 3, 8, and 16 months of age using photoreceptor, horizontal, and bipolar cell markers. Sixteen-month-old C57BL/6 mice retinas were used as controls. RESULTS: The DBA/2J mice had outer retinal degeneration at all ages, with the most severe degeneration in the oldest retinas. At 3 months of age, the number of photoreceptor cells and the thickness of the OPL were reduced. In addition, there was a loss of horizontal and ON-bipolar cell processes. At 8 months of age, RGC degeneration occurred in patches, and in the outer retina overlying these patches, cone morphology was impaired with a reduction in size as well as loss of outer segments and growth of horizontal and bipolar cell processes into the outer nuclear layer. At 16 months of age, connectivity between photoreceptors and horizontal and bipolar cell processes overlying these patches was lost. CONCLUSIONS: Retinal degeneration in DBA/2J mice includes photoreceptor death, loss of bipolar and horizontal cell processes, and loss of synaptic contacts in an aging-dependent manner.

The RNA binding protein RBPMS is a selective marker of ganglion cells in the mammalian retina.

There are few neurochemical markers that reliably identify retinal ganglion cells (RGCs), which are a heterogeneous population of cells that integrate and transmit the visual signal from the retina to the central visual nuclei. We have developed and characterized a new set of affinity-purified guinea pig and rabbit antibodies against RNA-binding protein with multiple splicing (RBPMS). On western blots these antibodies recognize a single band at 〜24 kDa, corresponding to RBPMS, and they strongly label RGC and displaced RGC (dRGC) somata in mouse, rat, guinea pig, rabbit, and monkey retina. RBPMS-immunoreactive cells and RGCs identified by other techniques have a similar range of somal diameters and areas. The density of RBPMS cells in mouse and rat retina is comparable to earlier semiquantitative estimates of RGCs. RBPMS is mainly expressed in medium and large DAPI-, DRAQ5-, NeuroTrace- and NeuN-stained cells in the ganglion cell layer (GCL), and RBPMS is not expressed in syntaxin (HPC-1)-immunoreactive cells in the inner nuclear layer (INL) and GCL, consistent with their identity as RGCs, and not displaced amacrine cells. In mouse and rat retina, most RBPMS cells are lost following optic nerve crush or transection at 3 weeks, and all Brn3a-, SMI-32-, and melanopsin-immunoreactive RGCs also express RBPMS immunoreactivity. RBPMS immunoreactivity is localized to cyan fluorescent protein (CFP)-fluorescent RGCs in the B6.Cg-Tg(Thy1-CFP)23Jrs/J mouse line. These findings show that antibodies against RBPMS are robust reagents that exclusively identify RGCs and dRGCs in multiple mammalian species, and they will be especially useful for quantification of RGCs.

Expression of voltage-gated calcium channel α(2)δ(4) subunits in the mouse and rat retina.

High-voltage activated Ca channels participate in multiple cellular functions, including transmitter release, excitation, and gene transcription. Ca channels are heteromeric proteins consisting of a pore-forming α(1) subunit and auxiliary α(2)δ and ß subunits. Although there are reports of α(2)δ(4) subunit mRNA in the mouse retina and localization of the α(2)δ(4) subunit immunoreactivity to salamander photoreceptor terminals, there is a limited overall understanding of its expression and localization in the retina. α(2)δ(4) subunit expression and distribution in the mouse and rat retina were evaluated by using reverse transcriptase polymerase chain reaction, western blot, and immunohistochemistry with specific primers and a well-characterized antibody to the α(2)δ(4) subunit. α(2)δ(4) subunit mRNA and protein are present in mouse and rat retina, brain, and liver homogenates. Immunostaining for the α(2)δ(4) subunit is mainly localized to Müller cell processes and endfeet, photoreceptor terminals, and photoreceptor outer segments. This subunit is also expressed in a few displaced ganglion cells and bipolar cell dendrites. These findings suggest that the α(2)δ(4) subunit participates in the modulation of L-type Ca(2+) current regulating neurotransmitter release from photoreceptor terminals and Ca(2+)-dependent signaling pathways in bipolar and Müller cells.

Neuronal connexin-36 can functionally replace connexin-45 in mouse retina but not in the developing heart.

The gap junction protein connexin-45 (Cx45) is expressed in the conduction system of the heart and in certain neurons of the retina and brain. General and cardiomyocyte-directed deficiencies of Cx45 in mice lead to lethality on embryonic day 10.5 as a result of cardiovascular defects. Neuron-directed deletion of Cx45 leads to defects in transmission of visual signals. Connexin-36 (Cx36) is co-expressed with Cx45 in certain types of retinal interneurons. To determine whether these two connexins have similar functions and whether Cx36 can compensate for Cx45, we generated knock-in mice in which DNA encoding Cx45 was replaced with that encoding Cx36. Neuron-directed replacement of Cx45 with Cx36 resulted in viable animals. Electroretinographic and neurotransmitter coupling analyses demonstrated functional compensation in the retina. By contrast, general and cardiomyocyte-directed gene replacement led to lethality on embryonic day 11.5. Mutant embryos displayed defects in cardiac morphogenesis and conduction. Thus, functional compensation of Cx45 by Cx36 did not occur during embryonic heart development. These data suggest that Cx45 and Cx36 have similar functions in the retina, whereas Cx45 fulfills special functions in the developing heart that cannot be compensated by Cx36.

A novel type of interplexiform amacrine cell in the mouse retina.

Mammalian retinas comprise an enormous variety of amacrine cells with distinct properties and functions. The present paper describes a new interplexiform amacrine cell type in the mouse retina. A transgenic mouse mutant was used that expressed the gene for the enhanced green fluorescent protein (EGFP) instead of the coding DNA of connexin45 in several retinal cell classes, among which a single amacrine cell population was most prominently labelled. Staining for EGFP and different marker proteins showed that these amacrine cells are interplexiform: they stratify in stratum S4/5 of the inner plexiform layer and send processes to the outer plexiform layer. These cells were termed IPA-S4/5 cells. They belong to the group of medium-field amacrine cells and are coupled homologously and heterologously to other amacrine cells by connexin45. Immunostaining revealed that IPA-S4/5 cells are GABAergic and express GAT-1, a plasma-membrane-bound GABA transporter possibly involved in non-vesicular GABA release. To characterize the light responses of IPA-S4/5 cells, patch-clamp recordings in retinal slices were made. Consistent with their stratification in the ON sublamina of the inner plexiform layer, cells depolarized in response to light ON stimuli and transiently hyperpolarized in response to light OFF. Responses of cells to green (578 nm) and blue (400 nm) light suggest that they receive input from cone bipolar cells contacting both M- and S-cones, possibly with reduced S-cone input. A new type of interplexiform ON amacrine cell is described, which is strongly coupled and uses GABA but not dopamine as its neurotransmitter.

ZO-1 and the spatial organization of gap junctions and glutamate receptors in the outer plexiform layer of the mammalian retina.

Information processing in the retina starts at the first synaptic layer, where photoreceptors and second-order neurons exhibit a complex architecture of glutamatergic and electrical synapses. To investigate the composition of this highly organized synaptic network, we determined the spatial relationship of zonula occludens-1 (ZO-1) with different connexins (Cx) and glutamate receptor (GluR) subunits in the outer plexiform layer (OPL) of rabbit, mouse, and monkey retinas. ZO-1 is well known as an intracellular component of tight and adherens junctions, but also interacts with various connexins at gap junctions. We found ZO-1 closely associated with Cx50 on dendrites of A-type horizontal cells in rabbit, and with Cx57 at dendro-dendritic gap junctions of mouse horizontal cells. The spatial arrangement of ZO-1 at the giant gap-junctional plaques in rabbit was particularly striking. ZO-1 formed a clear margin around the large Cx50 plaques instead of being colocalized with the connexin staining. Our finding suggests the involvement of ZO-1 in the composition of tight or adherens junctions around gap-junctional plaques instead of interacting with connexins directly. Furthermore, gap junctions were found to be clustered in close proximity to GluRs at the level of desmosome-like junctions, where horizontal cell dendrites converge before invaginating the cone pedicle. Based on this distinct spatial organization of gap junctions and GluRs, it is tempting to speculate that glutamate released from the photoreceptors may play a role in modulating the conductance of electrical synapses in the OPL.

Rod and cone contributions to horizontal cell light responses in the mouse retina.

Mammalian B-type horizontal cells make contact with both photoreceptor types: the dendrites contact cone photoreceptors, whereas the axon terminal processes contact rods. Despite their distinct synaptic contacts, horizontal cell somata and axon terminals receive a mixture of rod and cone inputs. Interaction of the two photoreceptor systems is essential for adaptation of photoreceptor sensitivity to different levels of background illumination, and horizontal cells play a key role in this adaptation. In this study, we used transgenic mouse lines to examine the contributions of rod and cone photoreceptor inputs to horizontal cell light responses in the mouse retina: rod signals were isolated by recording intracellularly from horizontal cells in a mouse lacking the cone cyclic nucleotide-gated channel, which lacks cone function, and cone signals were assessed using the rhodopsin knock-out mouse, which is a model for pure cone function. We found that both horizontal cell compartments receive a mixture of inputs from both photoreceptor types. To determine whether these inputs arrive via the long axon connecting the compartments or by way of rod-cone gap junctional coupling, we assessed the rod and cone contributions to horizontal cell somatic and axon terminal light responses in the connexin36-deficient mouse retina, which lacks rod-cone coupling. Our results confirm that rods and cones are coupled by connexin36, and suggest that signal transmission along the axon is unidirectional: signals are passed from horizontal cell soma to axon terminal but not from axon terminal to soma.